DESCRIPTION (From the Applicant's Abstract): Programmed cell death (apoptosis) occurs in virtually all organisms and is used to remove superfluous cells during normal development. A variety of stimuli can induce the cell death program and many molecules involved in the process have been identified. Three Drosophila genes, reaper, grim, and hid, trigger apoptotic cell death in a number of different contexts, yet the mechanism by which they act is unknown. We observed that N-terminal synthetic peptides derived from Reaper and Grim proteins induce inactivation of voltage-gated K+ channels in the same concentration range as the native inactivation particle and full-length Reaper protein results in stable K+ channel block at very low concentration. In essence, these molecules appear to be acting as stable K+ channel inactivation particles or intracellular K+ channel toxins. Conversely, application of full-length Reaper protein to the cytoplasmic side of Na+ channels leads to a decreased rate of Na+ channel inactivation. These observations have led to the following hypothesis: the apoptosis proteins, either through increased K+ channel inactivation or block and/or decreased Na+ channel inactivation, result in severe membrane depolarization and activation of the cell death cascade. The experiments described here are designed to test this hypothesis by examining the effects of wild-type and mutant Reaper proteins on K+ and Na+ channel activity, membrane potential, and cell killing activity in stably transfected PC12 cell lines. We will determine if Reaper-induced K+ channel block (or Na+ channel opening), membrane depolarization, and cell death can be prevented by over-expression of a Reaper-insensitive K+ channel, by using pharmacological agents that block Na+ channels, and/or by co-expression of other apoptotic inhibitors. Physical interactions between Reaper and K+ or Na+ channels in cells undergoing apoptotic cell death will be determined using co-immunoprecipitation assays. Finally, the effects of wild-type and mutant Reaper expression on cell killing activity in infected superior cervical ganglion sympathetic neurons will be examined to determine if our proposed mechanism for the initiation of apoptosis is true for primary neurons. This application integrates classical molecular genetic and cell biological methods for studying cell death with an electrophysiological approach that is expected to provide new insights into mechanisms of initiation of cell death and may contribute to the development of novel therapeutic strategies for the prevention or treatment of neurodegenerative disorders.